Mass production of foreign proteins using microorganisms is the most important technical area in protein pharmaceutical industry. In order to recover and purify the desired products, recombinant foreign proteins may be expressed intracellularly or otherwise may be secreted extracellularly. Upon intracellular expression of protein overexpression of the proteins may frequently lead to intracellular accumulation of proteins in the non-active and water-insoluble form in many cases and may bring about various disadvantages of productivity-lowering factors such as cumbersome processes to disrupt solid microorganisms and complicated and difficult purification processes to separate the desired protein from various kinds of host proteins present in the cells. Whereas, extracellular secretion of the desired proteins may provide easy way to avoid difficulties and problems associated with former intracellular expression of proteins. Further, since extracellular protein secretion can be successfully achieved only after correct folding and modification of the protein following the transcription process, protein production via extracellular secretion provides a benefit capable of obtaining an active form of a soluble protein having a correct tertiary structure. Therefore, it can be said that extracellular secretion of the proteins is superior to an intracellular accumulation system of recombinant foreign proteins, in terms of protein production yield as well as protein quality control.
However, in many cases, extracellular protein secretion system in a recombinant protein expression using a strong promoter frequently suffers from significantly low levels of expression and secretion, as compared to the intracellular accumulation system. In order to overcome such disadvantages and problems of extracellular secretion, a great deal of researches have been made to enhance an extracellular secretion yield of proteins. Most of researches have focused on direct optimization of signal sequence for effective secretion of protein or finding a novel and strong secretion signal sequence (Nucleic Acids Res Suppl., 2003(3):261-2; and Biochem Cell Biol. 1993, 71:401-5).
As another approach, there have been made many studies to enhance secretion efficiency of the desired protein by facilitating the protein folding corresponding to an initial rate-limiting step upon overexpression of recombinant foreign proteins and preventing insoluble precipitation which may take place prior to extracellular secretion of the protein, via induction of overexpression of chaperones that assist in folding and water-solubilization of proteins, using molecular biology techniques (Robinson and Wittrup, Biotech. Prog., 11: 171, 1995; Robinson et al., Biotechnology, 12:381-384, 1994; and Wulfing and Plukthun, Mol. Microbiol. 12(5): 685-692, 1995).
The galactose promoter, conventionally used to induce expression of foreign recombinant proteins in yeast, is a strong inducible promoter utilizing galactose as an inducer. Even though strong induction of protein expression may be advantageous to augment intracellular accumulation or expression level of foreign proteins, this may lead to decreased secretion efficiency or cellular dysfunctions due to the occurrence of insoluble precipitation in cells at an early stage of secretion when it is desired to secrete the desired protein extracellularly. Such an event is also occasionally observed in recombinant expression systems such as E. coli expression systems and yeast expression systems. In order to overcome such shortcomings, various attempts have been made to lower an incubation temperature (Baneyx F., Curr. Opin. Biotechnol., 10: 411-421, 1999; and George Georgiou and Pascal, Curr. Opin. Biotechnol., 7: 190-197, 1996).
However, such a method of lowering the incubation temperature suffers from a disadvantage associated with increased production costs of recombinant foreign proteins due to a prolonged incubation period. Therefore, there is an urgent need in the art for the development of a method capable of enhancing secretion efficiency of recombinant foreign proteins via regulation of an activity of a galactose-inducible promoter without lowering of the incubation temperature.
To this end, based on the idea that the activity of the galactose-inducible promoter may be regulated by controlling availability of galactose in a host, the inventors of the present invention have confirmed that it is possible to improve the secretion efficiency of recombinant foreign proteins by using a mutant strain lacking a galactose permease gene involved in absorption of galactose, or by culturing the transformed yeast strain via fed-batch culture with co-feeding of a certain ratio of galactose with glucose that mediates catabolite repression. The present invention has been completed based on these findings.